In vitro model for HIV and other viral diseases

ABSTRACT

An in vitro model system for viral infection is comprised of a tissue block from adult tonsil or lymph node supported on a matrix which is flexible and porous and wherin the supported tissue block is cultured in a medium whose surface is congruent with the tissue block/matrix interface. The histoculture system can be used to screen for antiviral drugs and to monitor the course of viral diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.08/885,242, filed Jun. 30, 1997, now abandoned, which is a continuationof and claims the benefit of the priority date of U.S. patentapplication Ser. No. 08/753,638, filed Nov. 27, 1996, now abandoned,under 35 U.S.C. § 120. The disclosure of the above-describedapplications are incorporated herein by reference in their entirety.

TECHNICAL FIELD

The invention relates to a model system for studying the progress ofviral diseases and for assessing possible therapeutic and diagnosticprotocols. Specifically, the invention concerns three-dimensionalhistocultures of adult lymph nodes and tonsils which provide substratesfor infection, study, and discovering of new therapeutics anddiagnostics.

BACKGROUND ART

The problem of providing a suitable model system for studying theprogress of human immunodeficiency virus (HIV) and other viral diseasesis well known. No in vivo model in animals for HIV, for example, isavailable at this time. Standard in vitro primary cell cultures do notmimic the full cellular repertoire within the tissues of an organism,and do not provide appropriate supracellular organization. Thus, itwould be advantageous to have an in vitro model which more accuratelymimics the behavior of the cells in the context of the organism.

Rosenszweig, M. et al., Leukemia (1994) 8:Suppl.1, S163-S165 andBonyhadi, M. L. et al., AIDS Res Hum Retrovir (1995) 11:1073-1080 havecultured neonatal thymus as a tissue model for HIV pathogenesis.However, while these cultures support productive infection of HIV-1,they show thymocyte cytopathology and profile changes only afterinfection with macrophage-tropic but not lymphocyte-tropic strains. Thehistocultures of the present invention can be derived from adult tissuesand are thus more representative of the progress of infection.

Various methods for culturing tumor cells in three-dimensional cultureare known. Some of these are summarized in Leighton, J. et al., CancerRes (1957) 17:929-941. A system for culturing human tumors in vitro inthree dimensions was described by Freeman, A. E. and Hoffman, R. M.,Proc Natl Acad Sci USA (1986) 83:2694-2698. Furtheore, athree-dimensional skin culture which could be used to evaluate toxicityand the effect of compounds on hair growth was described in WO92/15700published Sep. 17, 1992.

Thus, applying techniques for three-dimensional histoculture, a modelsystem for viral infection, particularly HIV infection, has beenprovided by the present invention.

DISCLOSURE OF THE INVENTION

The invention is directed to a histoculture system wherein lymph node ortonsil tissue is supported in a three-dimensional, structurally faithfulsystem to serve as a model for viral infection, particularly HIVinfection. Thus, in one aspect, the invention is directed to ahistoculture system which is useful as an in vitro model for viralinfection which system comprises a flexible macromolecular, porousmatrix, and supported thereon, an integral macroscopic section of animallymph node tissue or tonsil tissue, said matrix immersed in a suitableculture medium wherein the surface of the medium is approximatelycongruent with the interface between the tissue and the matrix. Thesection of tissue is then infected with an amount of virus effective tomaintain growth of the virus.

In other aspects, the invention is directed to the in vitro histoculturesystem which is thus infected with a virus. In still other aspects, theinvention is directed to a method to chart the progress of viralinfection using the histoculture system of the invention and to methodsto identify therapeutic compounds and protocols effective against theinfection using the histoculture as a model, or as a diagnostic over thecourse of treatment administered to a subject.

The histoculture system of this invention can mount an immune response,as well as support infection by a virus, such as HIV. Such infection caninhibit the immune response, causing immunodeficiency in vitro.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the geometric configuration ofthe histocultures of the invention.

MODES OF CARRYING OUT THE INVENTION

The method employs a histoculture system which permits growth of intacttissue samples in three dimensions while providing for adequate supplyof nutrients and oxygen. The histoculture system employs a soft, porousmatrix which is, preferably, comprised of extracellular material, suchas collagen, polysaccharides, and the like. Suitable matrices aredescribed in Leighton, J. (1957), Freeman (1986) and WO92/15700, allcited above, and all of which are incorporated herein by reference.

Briefly, the matrix is soft and flexible and may indent upon placementof the tissue such that the edges of the matrix may contact the verticaledges of the tissue. The matrix provides a trabecular structure withinterstices suited for capillary action to deliver nutrients in theaqueous medium to the tissue supported on the matrix. Whileextracellular macromolecules are preferred, other materials that may beappropriate for the matrix include nylon, borosilicate, glass fiber, orpolypropylene. However, preferred are solids comprising one or moreorganic molecules or molecular aggregates which are those produced andsecreted by cells into the extracellular space and which serve, in vivo,as support, adhesive and framework for maintaining three-dimensionaltissue organization and function. These molecules includehigh-molecular-weight proteins and glycoproteins, such as collagen,laminin, fibronectin and the like as well as complex polysaccharides.

Several commercially available materials can be used, including thegelatinized pork skin known as GELFOAM™, Upjohn Company, Kalamazoo,Mich.; a composition comprising laminin, collagen, proteoglycan andantactin such as MATRIGEL™, Collaborative Research, Inc., Bedford,Mass.; and a specialized collagen produced from pig skin by HealthDesign Industries, Rochester, N.Y. Other materials which can be used arehomopolysaccharide sponges, such as those described by Leighton, J. etal., J National Cancer Inst (1951) 12:545-561. A combination of thesematerials can be used as well, such as a top layer of collagencontaining gel and a bottom layer of homopolysaccharide sponge.

Other suitable matrices can be prepared de novo provided that they arecapable of conducting medium to the supported material through capillaryaction and provide flexible support capable of maintaining mediumapproximately at the interface between the support and the tissue placedthereon.

The ratio of the tissue sample size to the support matrix dimensions isvariable; preferably the matrix is of larger top supporting surface areathan required to cover the entirety of the tissue sample. Multiplesamples can be placed on the same matrix; it is preferred that they donot touch. The vertical dimension of the tissue is such that thenutnents of the medium can be transported through the tissue when themedium is maintained at approximately the lower surface of the tissue asplaced on the support matrix.

In culture, the samples are placed on the matrix so that the interfacebetween the tissue and the matrix coincides approximately with thesurface of the medium. Thus, the matrix with the tissue sample isimmersed in a volume of medium sufficient to contact the matrix but notto completely cover the tissue.

The support matrix is preferably pretreated with the culture mediumbefore the tissue sample is placed. This serves to equilibrate thematrix with the medium. Generally, the matrix is cut to a predeterminedsize and soaked in the medium in a sterile container for a timesufficient to saturate and equilibrate the matrix, typically 4 hours at37° C.

The media employed can vary, but a typical medium would comprise Eagle'sMinimum Essential Medium (MEM) with 10% fetal bovine serum, 0.1 mMnonessential amino acids, and the antibiotics gentamicin (100 μg/ml) andcefotaxime (95 μg/ml). Other antibiotics may also be used.

The histocultures of the invention have been shown to support livingtissue for extended periods and can be used as model systems for viralinfection, particularly HIV infection. The progress of viral infectioncan be monitored in a variety of ways as is understood in the art. Forexample, at various time points after application of an effectiveamouiit virus to maintain viral growth, the tissues can be dissociatedinto single cells and the cells counted. Any method for counting thecells may be used, typically the cells are stained, either with anappropriate dye or with a labeled antibody. The cells can then becounted using any art-recognized techniques, for example, by flowcytometry.

Alternatively, or in addition, the progress of infection can be assessedby other means such as assays designed to monitor surface cell markersor assays designed to distingish living from dead cells or assays whichare relevant to aspects of intracellular metabolism.

The histoculture system can mount an immune response to an addedantigen. This characteristic makes the system particularly valuablesince infection by HIV may inhibit this immune response, and the abilityof HIV infection to do so can be monitored. This provides an additionalmethod for screening protocols and compositions useful in treating HIV,since the system can be tested in the presence and absence of theprotocol or composition under conditions where an antigen has also beensupplied and the effect of the protocol or composition on thedevelopment of immunodeficiency can be measured.

For use in drug and protocol testing, the histocultures, infected withthe virus of interest, are cultured in the presence and absence of acandidate drug or protocol and the effect on the growth of the viralcells and on the health of viral-infected cells is assessed. Thus, ifviral growth is impaired in the presence of the drug or protocol ascompared to its absence, or if the cells maintain a healthy condition inthe presence of the drug or protocol as compared to absence thereof, thedrug or protocol is a successful candidate for viral treatment. Thisaspect is particularly important with respect to HIV, in view of thelack of suitable model systems for this virus. As stated above, thisassay system can also be modified when HIV is the infective agent bymonitoring the progress of immunodeficiency characterizing a response toan antigen in the presence of HIV infection. The effect of a candidateprotocol or composition in controlling this immunodeficiency responsecan then be assessed. The histocultures may also be used to cultureviruses of interest that may be difficult to passage in in vitroculture. In addition, the histocultures serve as diagnostic tools inassessing the progress of therapy. In this application, biopsies areremoved from a patient being treated with a therapeutic regime and theeffect on further growth in histoculture is assessed. This can beperformed at various stages of the disease.

The following examples are intended to illustrate but not to limit theinvention.

EXAMPLE 1 Preparation of Tonsil Histocultures

Human tonsils, surgically removed during therapeutic tonsillectomy, weredissected and blocks of tissue of about 1-2 mm in diameter were culturedon collagen sponge gel supports at a liquid/air interface for 10-26days, as described by Freeman, A. E. et al., Proc Natl Acad Sci USA(1986) 83:2694-2698.

The histocultures maintained their morphology, and extended networks offollicular dendritic cells were found inside germinal centers by 3Dreconstruction of confocal optical sections of histoculture blocksimmunostained with FITC-labeled anti-CD21 antibodies. The cultures alsoproduced IgG. Immunohistochemical analysis revealed well-definedgerminal centers formed by B cells with T cells concentrated aroundthem. Although in the first 24-36 hours, some depletion of lymphocytesoccurred, all of the key elements of tissue architecture were preservedeven in the fourth week in culture.

EXAMPLE 2 Virus Production

The histocultures prepared as in Example 1 were infected with HIV-1 at amultiplicity of infection (MOI) of 400-900 TCID₅₀ (median tissue cultureinfectious dose) per block. The strains used were as follows: LAV.04,(SI), a laboratory strain that induces syncytia in various cell linesand peripheral blood cells;

SF162, (NSI), a laboratory strain that does not induce syncytia;

Primary patient isolate 302144, (NSI), which does not induce syncytia;and

Primary patient isolate 302076, (NSI), which induces syncytia.

The latter two strains are from the NIH AIDS Research and ReferenceReagent Program.

In all cases, viral particle production started between days 5 and 6after infection. The amount of p24 measured by HIV-1 p24 antigen ELISAin the culture media increased exponentially until day 8, when itreached a plateau lasting until day 13. In culture media, virusinfectious titers increased from undetectable levels on day 1 afterinfection, to 10⁵ TCID₅₀ ml⁻¹ on days 8-10 after infection. Productiveviral infection was also confirmed by in situ hybridization with HIVRNA, using an antisense probe cocktail complementary to the entire HIVgenome (sensitivity of about 20 copies of RNA) and byimmunohistochemical detection of p24. By these criteria, no more than 3%of the tissue cells were productively infected.

No stimulation was required for the foregoing with either PHA or IL-2,in contrast to HIV infection in cultures of peripheral blood mononuclearcells.

Similar results were obtained when adult lymph nodes from recentlydeceased cadavers were substituted for tonsil tissue in the viralinfection model.

EXAMPLE 3 Monitoring Progress of Infection

Tissue blocks from the same tonsil were divided in half. One half wasinoculated with HIV. The response to infection was monitored bydissociating the tissue blocks into single cells at various time points,staining with fluorescent antibodies and counting cell populations usingflow cytometry.

Isolate-dependent depletion of CD4⁻ T cells in infected histocultureswas found. The earliest decline was noticed on day 4 after infection. Ondays 10-13, when virus production for isolates equalized for all typesof virus used and reached a plateau level, the CD4⁻/CD8⁻ ratio intissues infected with SI types LAV or patient isolate 302076 was lessthan 4% of the uninfected control. In histocultures infected with witherNSI type SF162 or primary isolate 302144, the CD4⁻/CD8⁻ ratio was only70% of the uninfected control. The CD4⁻/CD8⁻ ratio for blocks infectedwith the NSI isolate 302144 remained constant at day 20.

In addition to measuring relative cell populations, the absolute numbersof cell subsets in a block can be estimated by adding an internalstandard number of fluorescent beads to the dissociated cellsuspensions. On day 13 after infection with LAV, there was a totaldecline in the number of T lymphocytes (CD3⁻) to 40±3% of control. Therewas no statistically significant change in the CD8⁻ subset of cells. Theobserved decrease in the number of T cells was mainly due to depletionof the CD4⁻ subset to 4±2% of control.

What is claimed is:
 1. A method to identify a compound or protocol forthe treatment of viral infection or virally-induced immunodeficiency,which method comprises maintaining a histoculture system in the presenceand absence of said compound or protocol, monitoving the progress ofviral infection or virally-induced immunodeficiency in the presence andabsence of said compound or protocol; and comparing the progress ofinfection or virally-induced immunodeficiency in the presence andabsence of said compound or protocol, whereby an inhibition of viralgrowth or improvement in immune status in the presence as opposed to theabsence of said compound or protocol indicates effectiveness of thecompound or protocol in inhibiting viral growth or amelioratingvirally-induced immunodeficiency; wherein said histoculture systemcomprises a flexible macromolecular, porous matrix having a trabecularstructure, and supported thereon, an integral macroscopic section ofanimal lymph node tissue, said tissue having been infected with anamount of virus effective to maintain growth of the virus, said matriximmersed in a suitable culture medium wherein the surface of the mediumis approximately congruent with the interface between the tissue and thematrix, whereby the tissue architecture is preserved.
 2. The method ofclaim 1 wherein said virus is human immunodeficiency virus (HIV).
 3. Themethod of claim 1, wherein the matrix is an extracellular macromolecularmatrix.
 4. The method of claim 3, wherein the matrix is collagen matrix.